In the past years, the field of proteomics experienced a rapid expansion of innovative technology. As the range of protein expression and modification is dynamic and the complexity of the proteome is increased by post translational changes such as glycosylation and phosphorylation, improved methods for separation and characterization of specific proteins is desired for proteomics application.
Affinity surfaces are typically used for sample fractionation and purification of complex biological samples for the purpose of protein profiling and biomarker discovery. Surface enhanced laser desorption ionization (SELDI) is currently the most widely used mass spectrometric technique which couples protein separation directly to the mass spectrometer (Tang et al, Mass Spectrom Rev. 2004 January-February; 23(1):34-44).
The first dimension of protein separation uses chromatographic substrates which included anion exchange, cation exchange, normal phase, reverse phase and IMAC etc. The SELDI technique was developed to obtain clinical profile of biological fluids (for example, Serum plasma etc) and got attention when several studies shown promising potential in identifying potential biomarker or protein patterns with diagnostic value (Bertucci et al, Mol Cell Proteomics. 2006 October; 5(10):1772-86). This technique is currently implemented in protein chip system and various types of activated surfaces are commercially produced by Ciphergen Biosystem Inc (Tang et al, 2004, Pathol Res Pract. 2004; 200(2):83-94).
However, there are technical limitations of SELDI mass spectrometric technique in terms of both sensitivity and reproducibility and also the high cost of protein chip arrays (Ivanov et al, Proteomics. 2006 March; 6(5):1399-414). So there is clear need of high throughput proteomic approach such as novel single platform for sample separation, enrichment and subsequent MALDI time-of-flight (TOF) mass spectrometric analysis.
MALDI mass spectrometry has become convenient and reliable analytical tool for the analysis of proteins and peptides (Aebersold and Mann, Nature. March 13; 422(6928):198-207, 2003). In the MALDI mass spectrometry, a sample plate having an affinity surface is generally used for absorption of specific proteins or peptides.
Previously, poly (acrylic acid) immobilized on gold MALDI probe was derivatized with polyethyleneimine and used as a cationic surface for the absorption of phosphopeptides (Xu et al, Anal Chem. 2003 Jan. 15; 75(2):185-90). Derivatization of patterned poly (acrylic acid) films with polyethyleneimine also yields polycationic surfaces that adsorb DNA (Anal Chem. 2004 Jun. 1; 76(11):3106-11). Branched polyethyleneimine was also used to prepare protein resistant surfaces to obtain nonfouling surface properties with respect to avidin adsorption (Erol et al, Langmuir. 2006 Dec. 19; 22(26):11329-36). Phenylboronic acid has been used in affinity chromatography because of its ability to form covalent complexes with glycoproteins in complex protein mixtures (Liu and Scouten, J Mol Recognit. 1996 September-December; 9(5-6):462-7). The present inventors have previously shown the usefulness of immobilized aminophenylboronic acid on magnetic beads for the enrichment and detection of glycoprotein and glycated insulin by MALDI TOF mass spectrometry (Lee et al, J Am Soc Mass Spectrom. 2005 September; 16(9):1456-60, Farah et al, Biochim Biophys Acta. 2005 Oct. 10; 1725(3):269-82). More recently, phenylboronic acid modified hydrogel affinity chips were used for mass spectrometric analysis of glycated proteins (Gontarev et al, 2007).
However, characterizing glycoprotein is challenging because of the inherent heterogeneous and diverse nature of glycans and complexity of this modification (Raman et al, Nat Methods. 2005 November; 2(11):817-24). Glycosylation is the most common form of post-translational modification and it is estimated that 50-60% of proteins in the human body is modified by this process. Overall, glycosylated proteins represent the majority of cell surface markers and secreted proteins (Hagglund et al, J Proteome Res. 2004 May-June; 3(3):556-66). It has been documented that degree and type of glycosylation might be linked to the certain disease and could be utilized as markers for diagnosis purpose (Duarnd, Clin Chem. 2000 June; 46(6 Pt 1):795-805). The ability to efficiently and comprehensively profile glycoproteins in biological samples of interest is critical to many biological and clinical researchers.
Few reports are appearing recently, describing different approaches for preparation of modified sample plate for the detection and analysis of phosphoproteins/peptides and glycated proteins by the MALDI mass spectrometry (Shen et al, Anal Biochem. 2005 Oct. 15; 345(2):258-69; Dunn et al, Anal Chem. 2006 Mar. 1; 78(5):1574-80; Gontarev et al, Rapid Commun Mass Spectrom. 2007; 21(1):1-6).